How leachates from wasted cigarette butts influence aquatic life? A case study on freshwater mussel Anodontites trapesiali.

There are several reports on the damage smoking causes to human health available in the literature, but little is known about the environmental and biological consequences from inappropriate cigarette butt (CB) disposal in urban and natural environments. The immunotoxic, morphotoxic and mutagenic potential of leachates from cigarette butts (LCB) diluted at environmentally relevant rates (LCB1x: 1.375%; LCB10x: 13.75%) was evaluated in adult representatives of the bivalve species Anodontites trapesialis, which was adopted as model organism. Type II hyalinocytes and granulocytes (phagocytic cells) frequency increased in the hemolymph of subjects exposed to the pollutant for 14 days. Based on this outcome, LCB chemical constituents did not induce immunotoxic effects. The treatments also did not seem to have any impact on the subjects' hemocitary morphometry parameters: diameter, area, perimeter, circularity and nucleus - cytoplasm ratio. However, subjects in groups LCB1x and LCB10x recorded a larger number of hyalinocytes with some nuclear abnormality such as micronucleus, blebbed nucleus, asymmetric constriction nucleus, and nuclear multilobulation and binucleation. The association between these abnormalities and the treatments was confirmed by the Cr, Ni, Pb, Zn, Mn and Na bioaccumulation in tissue samples of the bivalve models exposed to LCB. To the best of our knowledge, this is the first report on LCB mutagenicity in representatives of a freshwater bivalve group. Given the chemical complexity of the addressed pollutants, it is imperative to develop further investigations about the topic.

First report on the mutagenicity and cytotoxicity of Zno nanoparticles in reptiles.

Understanding how human activities affect animal biodiversity is essential to investigations about the biological effects of several pollutants and contaminants dispersed in the environment. This is the case of zinc oxide nanoparticles (ZnO NPs), which are emerging pollutants whose effect on reptiles' health is completely unknown. Thus, the objective of the present study is to evaluate the possible damages induced by these NPs in Podocnemis expansa juveniles (Amazon turtle) by using morphological changes of circulating erythrocytes as nuclear toxicity biomarker. The animals were exposed to the intramuscular administration of 440 µg/kg and 440,000 µg/kg of ZnO NPs, for 10 consecutive days. The micronuclei assay and other nuclear abnormalities were performed at the end of the experiment, as well as different morphometric measurements applied to the erythrocytes. Based on the current data, ZnO NPs induced nuclear abnormalities such as micronuclei and binucleation, which are associated with carcinogenic processes and with flaws in the mitotic machinery. The low "nuclear area: erythrocyte area" ratio and larger cytoplasmic area observed for animals exposed to NPs evidenced erythrocytic change induction likely related to negative energy balance/metabolism interferences and/or to oxygen transportation efficiency by erythrocytes. This is the first report on the mutagenic and cytotoxic effect induced by NPs on representatives of a group of reptiles. This outcome suggests that further investigations must focus on better understanding the (eco)toxicological potential of ZnO NPs.

Behavioral toxicity of tannery effluent in zebrafish (Danio rerio) used as model system.

The ecotoxicity of untreated tannery effluent (UTE) in several animal models has been reported; however, its effects on fish behavior, and neurotoxicity, remain unknown. Thus, the hypothesis that the chronic exposure to UTE can induce behavioral changes in adult zebrafish (Danio rerio) representatives, even when it is highly diluted in water, was tested. Animals exposed to 0.1% and 0.3% UTE for 30 days showed behavioral changes in visual social preference tests through their co-specific and antipredator defensive responses, which had indicated neurotoxic actions. Zebrafish exposed to UTE appeared to have not co-specific preference when it is paired with Poecilia sphrenops. In addition, only animals in the control group showed aversive behavior in the presence of the herein used predatory stimulus (Oreochromis niloticus). However, Cr, Na and Mg bioaccumulation was higher in zebrafish exposed to 0.1% and 0.3% UTE, although anxiogenic and anxiolytic effects were not observed in the models exposed to UTE in the novel tank diving or aggressiveness-increase-in-the-mirror tests. This outcome allowed associating the exposure to the pollutant and bioaccumulation with the observed behavioral changes. The present study is pioneer in scientifically evidencing the sublethal impact caused by chronic exposure to UTE in experimental environment simulating realistic aquatic pollution conditions. Accordingly, results in the current research should motivate further investigations to broaden the knowledge about the real magnitude of UTE biological impacts on the aquatic biota.

Cigarette butt leachate as a risk factor to the health of freshwater bivalve.

The toxicity caused by smoking to human health has been demonstrated in several scientific studies. However, little attention has been given to damages caused to aquatic biota when cigarette butts (CB) are disposed of on water surface. Thus, the main aim of the current study is to evaluate the behavioural toxicity of cigarette butt leachates (CBL) in freshwater bivalve species Anodontites trapesialis exposed to different environmentally-relevant dilutions (CBL1x = 1.375%, CBL10x: 13.75%). There were significant CBL effects on the burrowing performance of the evaluated bivalves, after 14 exposure days. Animals exposed to CBL presented higher latency to foot emission and to start the burrowing process, as well as larger number of cycles required for burial. In addition, there were lower burrowing angle and burrowing rate index in CBL-exposed bivalves than in the unexposed ones. Chemical analyses performed on the muscle tissues of animals exposed to both CBL dilutions evidenced the bioaccumulation of several metals at high concentrations in CBL (Cr, Ni, Pb, Mn, Zn and Na); this outcome enabled associating these metals with behavioural changes observed in CBL-exposed groups. Thus, the current study firstly reports that even highly-diluted CBL concentrations can induce behavioural changes in freshwater bivalves, as well as that CBL extrapolation to natural environments can lead to several damages to the fitness of living organisms and to the dynamics of their population.

Ingestion of tannery effluent as a risk factor to the health of birds: A toxicological study using Coturnix coturnix japonica as a model system.

Tannery industries generate large amounts of tannery effluents (TE), which have been considered highly toxic to various groups of animals. However, the identification and characterization of the impact of this mix of pollutants on birds is still highly incipient. So, our goal was to evaluate the possible biological changes of Coturnix coturnix japonica, exposed for 45 days, to different dilutions of TE (1.4%, 3.1% and 6.5%), using behavioural biomarkers, mutagenics and egg production. When submitted to the behavioural tests, quails that ingested TE presented behaviour compatible with an anxiolytic effect in the open field test; absence of emotional reactivity in the object recognition test; reduced rates of predation of Tenebrio molitor larvae (potential prey); as well as an anti-predatory defensive response deficit when confronted, especially with Felis catus males (potential predator). In addition, we observed increased biomass of the liver, increased feed conversion index and lower feed efficiency index; mutagenic effect of TE (inferred by the increase of nuclear erythrocyte abnormalities); reduced productive performance and egg quality, in addition to different staining patterns of the eggs produced by quails from the control group. Therefore, our study confirms the toxicity of TE in C. coturnix japonica, even in small dilutions. While behavioural changes demonstrate the neurotoxic potential of the pollutant, the other alterations suggest that the mechanisms of action of its chemical constituents are not selective, that is, they act systemically, acting synergistic, antagonistic or additively, causing harmful effects in animals.

Analysis of ZnO nanoparticle-induced changes in Oreochromis niloticus behavior as toxicity endpoint.

The toxicity of zinc oxide nanoparticles (ZnO NPs) has been investigated in different animal models. However, concentrations tested in most studies are often much higher than the ones potentially identified in the environment. Therefore, such toxicity limits the application of these studies to evaluate ecotoxicological risks posed by these nanopollutants. Thus, the aim of the current study is to evaluate the impacts of ZnO NPs (at environmentally relevant concentrations - 760 µg/L and 76,000 µg/L, for 72 h) on the behavioral responses of Oreochromis niloticus (Nile tilapia) exposed to it. Results did not evidence harmful effects of NPs on animals' locomotor abilities (evaluated through open-field and light-dark transition tests), or anxiety-predictive behavior. On the other hand, Zn bioaccumulation in the body tissues of the analyzed tilapias was correlated to changes in eating behavior (motivated by ration pellets), as well as to deficits in antipredatory defensive behavior (under individual and collective conditions). Tilapia exposed to ZnO NPs recorded lower avoidance, flight and territorialist behavior rates when they were individually confronted with potential predators (Salminus brasiliensis). However, collectively exposed animals were unable to recognize their predators, as well as to differentiate them from artificial baits ("false predators"). The present study is the first to report biological impacts resulting from the short exposure of fish-group representatives to ZnO NPs. Thus, we believe that it may be relevant to improve the knowledge about ecotoxicological risks posed by these pollutants.

Do Amazon turtles exposed to environmental concentrations of the antineoplastic drug cyclophosphamide present mutagenic damages? If so, would such damages be reversible?

Antineoplastic drugs (AD) have been increasingly used, but the disposal of their wastes in the environment via hospital effluent and domestic sewage has emerged as an environmental issue. The current risks posed to these animals and effects of pollutants on the reptiles' population level remain unknown due to lack of studies on the topic. The aim of the present study was to evaluate the mutagenicity of neonate Podocnemis expansa exposed to environmental concentrations (EC) of cyclophosphamide (Cyc). The adopted doses were EC-I 0.2 µg/L and EC-II 0.5 µg/L Cyc. These doses correspond to 1/10 and » of concentrations previously identified in hospital effluents. Turtles exposed to the CyC recorded larger total number of erythrocyte nuclear abnormalities than the ones in the control group after 48-h exposure. The total number of abnormalities for both groups (EC-I and EC-II) 96 h after the experiment had started was statistically similar to that of animals exposed to high Cyc concentration (positive control 5 × 104 µg/L). This outcome confirms the mutagenic potential of Cyc, even at low concentrations. On the other hand, when the animals were taken to a pollutant-free environment, their mutagenic damages disappeared after 240 h. After such period, their total of abnormalities matched the basal levels recorded for the control group. Therefore, our study is the first evidence of AD mutagenicity in reptiles, even at EC and short-term exposure, as well as of turtles' recovery capability after the exposure to Cyc.

Anti-cancer drugs in aquatic environment can cause cancer: Insight about mutagenicity in tadpoles.

Cyclophosphamide (Cyc) and 5-fluorouracil (5-FU) are two of the most used antineoplastic drugs (AD) in the world. However, their discharge in the environment became a yet-unknown environmental issue that has impact on some groups of animals, such as amphibians. We assessed tadpoles (Lithobates catesbeianus) exposed to environmental concentrations (EC) of Cyc and 5-FU to evaluate whether they can cause morphological and mutagenic changes in them. We defined the following groups: control, positive control (50 mg/L of Cyc), EC-Cyc-I (0.2 µg/L), EC-Cyc-II (0.5 µg/L), EF-Cyc (2.0 µg/L), EC-5-FU-I (13.0 µg/L), EC-5-FU-II (30.4 µg/L) and EF-5-FU (123.5 µg/L). EC groups presented predictive AD concentrations in 10% and 25% hospital-effluent dilutions in water. EF groups met gross hospital-effluent concentrations. Based on our data, ADs caused intestinal changes and influenced the interocular distance in tadpoles after 30-day exposure. We also observed the aneugenic and clastogenic effect of ADs due to the higher frequency of micronucleated and binucleated erythrocytes, and blebbed, multilobulated, notched and kidney-shaped nuclei in animals exposed to them. Based on such changes, we assume that Cyc and 5-FU can trigger malignant cell transformation processes, and cancer, in animals exposed to them, even at low concentrations. Our study is the first to describe that Cyc and 5-FU, spread in the environment, cause damages in non-target organisms opposite to their original end.

The potential reproductive toxicity of tannery effluent to the estrous cycle and ovarian follicular dynamics of female Swiss mice.

Although the toxic effects of tannery effluent (TE) on tanning-industry workers have been reported in many studies, its effects on females' reproductive system are unknown. We aimed at evaluating the effects of direct contact with TE on the "emotional" status, estrous cycle (during 15 consecutive exposure days), and ovarian follicular dynamics of female Swiss mice at the end of the experiment to broaden the knowledge about the toxicity of this pollutant. The herein adopted exposure protocol simulated tanning-industry workers' exposure to TE. The test animals were subjected to 45 exposure days, for 1 h a day, 5 days a week (from Monday to Friday). Based on the collected data, female mice exposed to TE recorded high anxiety index in the elevated plus maze test, although we did not observe changes in their estrous cycle. The smaller total and specific number of ovarian follicles (types 1 to 6) and the higher frequency of degenerating follicles (atresic) in female mice exposed to TE marked the folliculogenesis reduction in them. Therefore, our study was the first to provide evidences that the exposure to TE can cause reproduction issues in female mice, as well as the first experimental insight about the impact of unhealthy work activities in tanning industries on women's reproductive system.

Mice exposure to haloxyfop-p-methyl ester at predicted environmentally relevant concentrations leads to anti-predatory response deficit.

Although the efficiency of haloxyfop-p-methyl ester (HPME) as selective herbicide is acknowledged, its impact on non-target organisms is poorly known. It is not known whether the short exposure of mammals to low HPME concentrations (consistent with a realistic contamination scenario) poses risks to these animals. Thus, the aim of the current study is to evaluate the effects of HPME on the anti-predatory behavior of female Swiss mice exposed to it. The animals were divided in groups: non-exposed (control) and exposed (route: i.p., for 2 days) to different herbicide concentrations (2.7 × 10-4 g/kg and 2.7 × 10-2 g/kg of body weight), which were considered environmentally relevant predicted concentrations. The animals were subjected to the open field and elevated plus-maze tests; results showed that the HPME did not lead to anxiolytic or anxiety behavior, or to locomotive changes in the tested animals, fact that was confirmed through the Basso Mouse Scale for locomotion scores. On the other hand, animals exposed to the herbicide were incapable of recognizing the snake as potential predator. Animals in the control group, exposed to a real snake (Pantherophis guttatus) remained longer in the safety zone of the test device, presented lower frequency of self-grooming behaviors for a shorter period-of-time, besides showing longer freezing time, which was not observed in animals exposed to HPME. Therefore, our study indicates the ecotoxicological potential of the herbicide, since anti-predatory behavior disorders may affect preys' responses and population dynamics.

Behavioral response and dynamics of Eisenia fetida hemocytes exposed to environmentally relevant concentration of sulfentrazone.

Although the toxicity of the pesticide sulfentrazone in some aquatic organisms is known, its effects on edaphic organisms such as earthworms remain completely unknown. Thus, we aimed at evaluating the behavior and immune response of Eisenia fetida exposed to sulfentrazone at environmentally relevant concentrations (EC). E. fetida representatives exposed to this contaminant (for 48 h) were divided in the following groups: environmental concentration (EC1x: 318 ng sulfentrazone/g of dry weight soil) and EC100x (concentration 100 times higher than in EC1x). Based on the avoidance test results, earthworms responded to this pesticide and proved the toxicity of sulfentrazone. The observed immune response induction was expressed by increased granulocytes presenting phagocytic vacuoles and agglomerations/encapsulations, mainly in animals belonging to groups EC1x and EC100x. However, the reduced frequency of plasmocytes in these animals' hemolymphs suggested that the phagocytic immune response was not efficient to assure 100% survival. Our study is the first to report sulfentrazone toxicity in an edaphic organism, at environmental concentration.

The effects of predicted environmentally relevant concentrations of ZnO nanoparticles on the behavior of Gallus gallus domesticus (Phasianidae) chicks.

The toxicity of ZnO nanoparticles (NPs) has been the subject of several investigations; however, concentrations much higher than the ones potentially found in the environment are often tested. In addition, groups of animals such as birds have not been used as model in studies in this field, fact that creates an important ecotoxicological gap in them. The aim of the present study is to investigate the effects of the exposure to environmentally relevant concentrations of ZnO nanoparticles on the anti-predatory behavior of chicks (Gallus gallus domesticus). The test animals were daily exposed to an environmentally relevant concentration of ZnO nanoparticles (0.245 mg kg-1) and to a toxic concentration of it (245.26 mg kg-1) through intraperitoneal injection for two days. We set a control group for comparison purposes. According to our results, ZnO nanoparticles did not affect the locomotor activity of, and did not cause anxiolytic or anxiogenic effect on, birds in the open field test. However, based on the lowest cluster score recorded during the social aggregation test, chicks exposed to ZnO nanoparticles failed to recognize the grunt of a hawk (Rupornis magnirostris) as predatory threat. Only birds in the control group recognized the test snake (Pantherophis guttatus) as potential predator. The higher Zn concentration in the brains of animals exposed to ZnO nanoparticles evidenced the capacity of these nanomaterials to cross the blood-brain barrier, even at low concentrations. This blood-brain barrier crossing could have affected the structures or neuronal mechanisms that modulate the defensive response of birds. Assumingly, even the minimal exposure to low concentrations of ZnO nanoparticles can affect birds. Our outcomes corroborate previous studies about the biological risks of water surface contamination by metal-based nanomaterials.

Mutagenic assessment of Lithobates catesbeianus tadpoles exposed to the 2,4-D herbicide in a simulated realistic scenario.

The aim of the current study is to assess possible erythrocyte mutagenic effects on Lithobates catesbeianus tadpoles exposed to water contaminated with 2,4-D. In order to do so, tadpoles were exposed to a predictive and environmentally relevant herbicide concentration (1.97 mg/L), which is likely to be found in lentic environments formed by superficial water runoffs in pasture areas where the herbicide was applied. The micronucleus test, as well as tests for other nuclear abnormalities, was conducted after 3, 5, and 9 days of exposure (d.e.). Changes in the biomass and mouth-cloaca length or interference in the larval development of the animals (in the three evaluated times) were not recorded. However, tadpoles exposed to 2,4-D showed the highest total number of nuclear abnormalities, as well as the highest frequency of binucleated erythrocytes and kidney-shaped nuclei (shortly after 3 d.e.). The micronucleus frequency was also higher in animals exposed to 2,4-D (in the 3rd, 5th, and 9th d.e.), as well as the frequency of binucleated cells (3rd, 5th, and 9th d.e.) presenting notched (9th d.e.) and blebbled (9th d.e.) nuclei in comparison to those of the control, after 5 and 9 days of exposure. Therefore, the current study is a pioneer in showing that 2,4-D has a mutagenic effect on L. catesbeianus tadpoles, even at low concentrations (environmentally relevant) and for a short period of time, a fact that may lead to direct losses in anuran populations living in areas adjacent to those subjected to 2,4-D herbicide application.

Organic waste vermicomposting through the addition of rock dust inoculated with domestic sewage wastewater.

The aims of the present study are to assess the organic waste vermicomposting process (cattle manure mixed with tannery sludge) by using inorganic waste (rock dust) inoculated with treated domestic wastewater sewage, as well as the vermicompost application in Ruellia brittoniana seedling production. Different proportions of organic and inorganic waste moistened (or not) in wastewater were vermicomposted (by Eisenia foetida) for 120 days in the first stage of the experiment. Statistically significant earthworm density increase was observed between the 60th and 90th experimental vermicompositing days in all the assessed groups. There was decreased E. foetida population density after 90 days. The K, P, TOC, C/N ratio and Ca, Na and Mg concentrations significantly decreased at the end of the vermicompositing process in comparison to the initial concentrations identified in most treatments. On the other hand, there was pH and N, Fe, Zn and Mn concentration increase in most of the vermicomposts assessed at the end of the experiment. All plants grown in soil containing vermicomposts presented higher Dickson Quality Index (DQI) than the control group, which was cultivated in soil containing commercial topsoil. Plants grown in soil containing 100% cattle manure and tannery sludge, moistened in treated domestic wastewater sewage, showed the highest DQI. Thus, the vermicomposting waste used in the present study, which was inoculated with treated domestic wastewater sewage, is an interesting vermicompost production technology to be used in ornamental plant production.